ID OF MOLECULES THAT SUBSTITUTE FOR ACTIVE NOTCH1 IN T LINEAGE LEUKEMOGENESIS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 NOTCH1编码一种对T细胞发育至关重要的跨膜受体，在约55%的人类T细胞急性淋巴细胞白血病(T-ALL)病例中发生突变。Notch1蛋白的激活需要被伽马分泌酶蛋白酶切割，从而释放Notch1胞内域(NICD)，然后移位到细胞核并上调转录。Notch1的突变形式是伽玛分泌酶依赖的，已经开发了几种伽玛分泌酶抑制剂(GSI)。我们利用Mx1-cre；LSL-KrasG12D小鼠中的逆转录病毒插入突变(RIM)产生了一大批在遗传和生化上不同的T细胞。在这个模型中，Notch1经常发生突变，使用识别Val 1477的特定抗体可以检测到NICD，Val 1477只有在伽马分泌酶切割后才暴露出来。我们在20个肿瘤来源的细胞系中彻底询问了Notch1的依赖性，发现对GSI的敏感性与NICD的存在相关，GSI治疗降低了NCID水平。这项工作目前正在PNAS上进行。有趣的是，我们鉴定了3个细胞株，它们强烈表达NICD特异性抗体识别的蛋白质，但对GSI具有抵抗力。这种蛋白质不会在接触GSI后消失。进一步的分析表明，这些株系不表达Notch1基因或上调了Notch1靶基因，表明Notch1没有被激活。重要的是，我们从未检测到这种蛋白质，并希望使用质谱学来确定其身份。未来的实验将包括：1)使用shRNA敲除T-ALL细胞中的蛋白质，以评估其在肿瘤发生中的作用；以及2)筛查人类T-ALL患者样本中编码该蛋白质的基因突变。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. NOTCH1 encodes a transmembrane receptor that is critical for T cell development and is mutated in ~55% of human T lineage acute lymphoblastic leukemia (T-ALL) cases. Activation of the Notch1 protein requires cleavage by the gamma-secretase protease, which releases the Notch1 intracellular domain (NICD) that then translocates to the nucleus and upregulates transcription. Mutated forms of Notch1 are gamma-secretase dependent and several gamma-secretase inhibitors (GSIs) have been developed. We harnessed retroviral insertional mutagenesis (RIM) in Mx1-cre; LSL-KrasG12D mice to generate a large panel of genetically and biochemically diverse T-ALLs. In this model, Notch1 is frequently mutated and NICD can be detected using a specific antibody that recognizes Val 1477 that is exposed only after gamma-secretase cleavage. We have thoroughly interrogated Notch1 dependence in 20 tumor-derived cell lines and found that sensitivity to GSI correlates with the presence of NICD and GSI treatment reduces NCID levels. This work is currently in press at PNAS. Interestingly, we identified 3 cell lines that robustly express a protein recognized by the NICD specific antibody, but are GSI-resistant. This protein does not disappear following GSI exposure. Further analysis revealed that these lines do not express Notch1 mRNA or have upregulation of Notch1 target genes, indicating that Notch1 is not activated. Importantly, we never detect this protein and would like to use mass spectrometry to determine its identity. Future experiments would involve 1) using shRNA to knockdown the protein in T-ALL cells to assess its role in tumorigenesis, and 2) screening human T-ALL patient samples for mutations in the gene that encodes this protein.